Previns as specific inhibitors and therapeutic agents for Botulinum toxin B and Tetanus neurotoxins

ABSTRACT

The compounds of the invention are generally described by the formula: 
 
B 1 Z* 2 B 3 Z* 4 X* 5 Q 6 F 7 X 8 X 9 X 10 X 11    (1), 
 
B 1 X 2 X 3 X 4 X 5 Q 6 F 7 X 8 X 9 X 10 X 11    (2), or 
 
B 1 Z 2 B 3 X 4 Z 5 Q 6 F 7 Z 8 X 9 X 10 X 11    (3) 
and the salts, esters, amides, and acyl forms thereof. Each position represented by a letter indicates a single amino acid residue: B is a basic of polar/large amino acid or a modified form thereof; X is a small or hydrophobic amino acid or a modified form thereof; X* is a small or polar/large amino acid or a modified form thereof; Z is a polar/large or hydrophobic amino acid or a modified form thereof; Z* is Proline or a polar/large of hydrophobic amino acid or a modified form thereof. As described below, one or more of the peptide linkages between the amino acid residues may be replaced by a peptide linkage mimic. These compounds may be used as molecular building blocks to create compounds that are optimized for inhibiting the protease activity of Botulinum b and tetanus toxins.

TECHNICAL FIELD

The invention relates to a class of peptide and peptide-like compounds,“Previns” which inhibit the enzymatic activity of Botulinum toxin B andTetanus neurotoxins and may be used as molecular building blocks forcreating compounds which are optimized for inhibiting the proteaseactivity of Botulinum toxin B and Tetanus neurotoxins.

BACKGROUND OF THE INVENTION

The Botulinum toxins (Bttxs) are among the most potent toxins toanimals, e.g. the LD₅₀ in mice is about 1 ng/kg. Bttxs comprise a familyof seven distinct serotypes (A-G). Bttxs are composed of two subunitscomprising a 100 kdal nerve-cell targeting heavy chain and a 50 kdalendoproteolytically active light chain. These toxins areZn-metalloproteases and contain a Zn-protein binding motif HEXXH.

However, Zn-metalloprotease inhibitors, such as angiotensin convertingenzyme inhibitors, captopril and phosphoramidon, are not effectiveinhibitors of Bttxs. Although Zn-chelators inhibit Bttx proteaseactivity in vitro, they merely delay the protease activity in vivo andin tissue preparations comprising intact nerve and muscles cells and/ortissues. Furthermore, some Zn-chelators are toxic at concentrationsnecessary to delay the Bttx protease activity. Although dithiocarbamatesinhibit other Zn-containing proteins such as SOD, they are ineffectiveagainst the Bttx serotype B (BttxB). Clearly, inhibitors of the variousBttx serotypes, such as BttxB, are needed.

BttxB specifically cleaves synaptobrevin (VAMP2) between glutamine 76and phenylalanine 77 (QF bond or cleavage site). There is an obligatoryrequirement for a relatively long substrate for the in vivo target VAMP2as shown by efforts to produce a minimum length substrate. It has beenshown that 30 amino acids of VAMP2 are required and 40 amino acids ofVAMP2 are required for optimum cleavage. See Shone, C. C. et al. (1993)Eur. J. Biochem. 217:965-971. V2, a peptide derived from VAMP2, is asequence of 10 amino acids located 4 residues upstream from the cleavagesite, and was found to inhibit Bttx activity. See Pellizzari R. et al.(1996) J. Biol. Chem. 271:20353-20358. In VAMP2, a mutation of theC-terminal amino acids had little effect; whereas a helix disruptingsubstitution of Pro for Ala inhibited BttxB activity by 28%. Further,replacement of several negatively charged amino acids led to almostcomplete inactivity. See Whitcome, M, et al. (1996) FEBS Let.386:133-136).

Computer-aided secondary structure analysis of VAMP2 predicted twostretches of α-helical structure flanking the cleavage site QF. SeeWitcome, M. R. et al. (1996) FEBS Let. 386: 133-136. Computer-aidedtertiary structure analysis indicates that the two helices could selfassociate to form a supersecondary structure of a helix bundle with thehelices separated by a reverse turn. See Lebeda F. J., et al. (1996)Med. Defense Biosci. Rev. 204.

The above results indicate that more than just the QF bond is requiredto be recognized by the toxin for substrate cleavage.

We have previously described a new class of compounds, Buforinins, thathave a characteristic conformation, a QF bond, and inhibit BttxBprotease activity.

Recently, however, we have elucidated core structures present in theseBuforinins and core structures exemplified by Substance P which mayserve as the foundation structure or molecular building block ofcompounds which inhibit the protease, activity of BttxB and Tttx. Thesecore sequences may be the elemental structure of compounds which inhibitthe protease activity of BttxB and Tttx. These core sequences and theiruses are disclosed herein below.

SUMMARY OF THE INVENTION

The invention is directed to the core structures called “Previns” whichhave an internal QF bond and the ability to inhibit BttxB proteaseactivity. As the tetanus toxin cleavage site is the same as BttxB, thecore structures may also serve as the core structures of compounds thatcompetitively inhibit tetanus protease activity.

Thus, in one aspect, the invention is directed to compounds having astructure of the formula:B₁Z*₂B₃Z*₄X*₅Q₆F₇X₈X₉X₁₀X₁₁   (1),B₁X₂X₃X₄X₅Q₆F₇X₈X₉X₁₀X₁₁   (2), orB₁Z₂B₃X₄Z₅Q₆F₇Z₈X₉X₁₀X₁₁   (3)and the salts, esters, amides, and acyl forms thereof. Each positionrepresented by a letter indicates a single amino acid residue: B is abasic or polar/large amino acid or a modified form thereof; X is a smallor hydrophobic amino acid or a modified form thereof; X* is a small orpolar/large amino acid or a modified form thereof, Z is a polar/large orhydrophobic amino acid or a modified form thereof; Z* is Proline or apolar/large or hydrophobic amino acid or a modified form thereof. Asdescribed below, one or more of the peptide linkages between the aminoacid residues may be replaced by a peptide linkage mimic.

In other aspects, the invention is directed to recombinant materialsuseful for the production of those peptides of the invention thatcontain gene-encoded amino acids, as well as plants or animals modifiedto contain expression systems for the production of these peptides. Theinvention also includes methods to prepare and manipulate theserecombinant materials.

In addition, the invention is directed to pharmaceutical compositionscontaining compounds, containing the core structure of the invention, asactive ingredients and to compositions which contain expression systemsfor the production of the peptides. The invention is also directed tomethods to prepare compounds containing the core structure of theinvention synthetically, to antibodies specific for these compounds, andto the use of the compounds as preservatives, therapeutics, andprophylactics.

The invention is also directed to the use of the compounds containingthe core structures of the invention in assays for detection of BttxBand Tttx by the use of selective inhibition and for determininginhibitors and substrates for a given toxin.

The present invention relates to materials, compositions, kits andmethods for inhibiting the enzymatic activity of Botulinum toxin B andTetanus neurotoxins. The invention further relates to materials,compositions, kits and methods for preventing or treating toxicpoisoning such as Botulinum toxin B and tetanus poisoning. The kits canprovide single or multiple dosage and can include other conventionalancillary materials such as instructions, solutions and compositionsneeded for operation. The compositions and solutions may be placed incontainers, test tubes, etc. Containers could be similar to thoseemployed in insect/snake bite kits that includes an injector whichprovides compounds containing the core structure of the invention andTCEP in separate chambers. A kit for determining whether a samplecontains a compound having the core structure of the invention, theamount of said compound or the type of said compound may includeantibodies immunospecific for the core structure.

A kit for determining whether a sample contains a Botulinum toxin or thetype of the Botulinum toxin may include antibodies immunospecific for atleast one compound containing the core structure having an interactionwith a Botulinum toxin. Likewise, a kit for determining whether a samplecontains a Tetanus toxin would include antibodies immunospecific for atleast one compound containing the core structure of the invention havingan interaction with a Tetanus toxin.

Another embodiment includes Buforin I along with one or more knownpeptide inhibitors associated with the decontamination of Botulinum Band/or Tetanus toxins. Additionally, the kits may also include a stablepeptide mixture or powder which includes compounds having the corestructures of the invention for sprinkling over food or wounds fordetoxification.

Yet another embodiment includes the use of the compounds of theinvention as molecular building blocks to construct compounds optimizedfor inhibition of the protease activity of BttxB and Tttx.

DESCRIPTION OF THE DRAWINGS

This invention is further understood by reference to the drawingswherein:

FIGS. 1A and B show that although Substance P is not a substrate ofBttxB, it is an inhibitor.

FIG. 2 shows the degree of BttxB inhibition of Substance P.

FIG. 3 shows a double reciprocal plot of inhibition of BttxBendoprotease activity by Buforin I.

FIG. 4 illustrates the inhibition of BttxB endoprotease activity byvarious Buforinins.

FIG. 5 illustrates the X-ray crystallographic structure of avianchromosomal protein histone octamer H2A residues Lys15-Try39 produced byBrookhaven Protein Database #1 HIO.

FIG. 6A is a comparison of the amino acid sequences of Buforin I, andmutant B-I R11L and mutant B-I R11L, K15L, S18L.

FIG. 6B shows helical wheel projections for Buforin I of Helix I andHelix 2.

FIG. 6C shows helical wheel projections for Helix 1 of mutants B-I R11Land B-1 R11L, K15L, S18L.

FIG. 7 shows typical compounds of the formula (1), (2) or (3).

DETAILED DESCRIPTION OF THE INVENTION

In our search for BttxB inhibitors, we investigated peptides thatcontain the QF cleavage site but are not identical in primary sequenceto VAMP2 surrounding the QF site. Substance P, an 11 amino acid peptidecontaining the QF bond is not a substrate of BttxB. See Example 1 and 2;and FIG. 1. This result supports the preferred helix-turn-helix and/orlong substrate hypothesis.

Buforin I (B-I) is a peptide isolated from the stomach of the Asian toadBufo bufo gargarizans which has a QF bond. Therefore, we used ourendopeptidase assay to determine if B-I is a substrate or an inhibitorof BttxB protease activity. We found that B-I is not a substrate forBttxB and that B-I dose-dependently and competitively inhibits BttxBactivity. See FIGS. 2 and 3. The extent of inhibition gave anIC₅₀=1×10⁻⁶ M. See FIG. 4. This was a surprising result as B-I is only18% homologous for conserved amino acids with VAMP2 55-94. See Table 1.TABLE I Sequence alignment of VAMP2, Buforin I and Buforin I derivativepeptides and Substance P Peptide Sequence VAMP2₅₅₋₉₄  ERDQKLSELDDRADALQAGASQFETSAAKLKRKYWWKNLK Buforin I^(a)AGRGKQGGKVRAKAKTRSSRAGLQFPVGRVHRLLRKGNY Buforin II^(b)               TRSSRAGLQFPVGRVHRLLRK Peptide24^(c)               TRSSRAGLQFPVGRVHRLLRKGNY Peptide36^(c)AGRGKQGGKVRAKAKTRSSRAGLQFPVGRVHRLLRK Sub P^(d)                  RPKPQQFFGLM^(a)Archer, B. T. III., et al. (1990) J. Biol. Chem. 265:17267-17273^(b)Park C. B., et al. (1996).^(c)Garcia, G. E. et al. (1998).^(d)Chang, M. M. et al. (1971) Nat. New Biol. 232:86-87.

We then evaluated truncated B-I peptides with our endopeptidase activityassay. The truncated peptides we evaluated are Peptide 36 which containsamino acids 1-36 of B-I and Peptide 24 which contains amino acids 16-39of B-I. Like B-I, these truncated peptides were not substrates of BttxB;however, the truncated peptides are less effective inhibitors of BttxBactivity as B-I. See FIG. 2. Peptide 36 was about 50% as effective asB-I. Peptide 24 was about 25% as effective as B-I. We also evaluatedBuforin II (B-II), which contains amino acids 16-36 of B-I, and foundthat B-II was 25% as effective as B-I.

B-I is derived from histone protein 2A (H2A) of the toad which is nearlyidentical to the sequence of avian H2A. See Table 2A and see Park, C.B., et al. (1996) Biochem. Biophys. Res. Comm. 218:408-413. Table 2Bshows the comparison of relevant amino acid sequences between SubstanceP and Buforin II. X-ray crystallographic analysis of the chicken histoneprotein particle shows that, for the region K15 to Y39, there arehelices upstream and downstream of the QF site. See FIG. 5 and SeeArents, G., et al. (1991) PNAS 88:10148-52 and Wang, S. W., et al.(1985) Nucleic Acids Res. 13:1369-138. Also, NMR analysis of B-II showsthat the region upstream from the QF site could form α-helix. See Yi, etal. (1996) FEBS Lett. 398:87-90. TABLE 2A H2A comparison of chicken totoad for relevant amino acid sequences Database^(GB) % Source Accessionno. Homology^(a) Bufo bufo gagarizans BBU70133GRGKQGGKVRAKAKTRSSRAGLQFPVGRVHRLLRKGNY Gallus gallus X02218GRGKQGGKARAKAKSRSSRAGLQFPVGRVHRLLRKGNY 100The suffix ^(GB) signifies accession numbers in the GenBank database.^(a)Homology to toad sequence. Similarity; basic: Arg, Lys; acidic: Asp,Glu; polar: Asn, Gln; hydrophobic: Ala, Ile, Leu, Met, Val; aromatic:Phe, Tyr, Trp; Hze. Ala, Ser, Thr.[1] Kim, H. S., Park, C. B., Kim, M. S., Kim, S. C. (96) biochem.Biophys. Res. Comm. 229:381-387.[2] Wang, S. W., Robins, A. J., d = Andrea, R. Wells, J. R. (85) NucleicAcids Res. 13:1369-1387.

TABLE 2B Comparison of the relevant amino acid sequences betweenSubstance P and Buforin II Database % Source Accession no. Homology^(a)Substance P P41333^(SP) RPKPQQFFGLM Buforin II BBU 736002.1^(GB)TRSSRAGLQFPVGRVHRLLRKThe suffix ^(SP) signifies accession numbers in the Swiss Proteindatabase.The suffix ^(GB) signifies accession numbers in the GeneBank database.

These results indicate that there is potential for long Buforinins toform a similar supersecondary structure of a reverse turn with helixbundling. See Table 3. Therefore, we have defined a new class ofpeptides, “Previns” which may be used to construct compounds such asBuforinins which includes Buforin I (39 amino acids), Buforin 11 (21amino acids), Peptide 36 and Peptide 24, and other analogous peptideshaving a QF bond, that competitively inhibit BttxB protease activity.TABLE 3 Computer-Aided Secondary Structure Prediction^(a) VAMP2₅₅₋₉₄ERDQKLSELDDRADALQAGASQFETSAAKLKRKYWWKNLK Gibrat^(b)HHHHHHHHHHHHHHHHHCCHHHHHHHHHHHHHHTTHHTCT Nnpredict^(c)--------HH-HHHHHHH---HHHHHHHHHHHHHHH---- B-IAGRGKQGGKVRAKAKTRSSRAGLQFPVGRVHRLLRKGNY GibratHTTTTTCCEEEEHHHHHHHHHCTEEEEHHHHEEEETTTC Nnpredict---------EHE-----------E----HHHHHHH---- B-I truncated 5 amino acids onboth ends QGGKVRAKAKTRSSRAGLQFPVGRVHRLL GibratHCCHHEEHHHHHHHHHCCEEEECHEHEEE Nnpredict --------E----HHHHHH-^(a)H, helix; E, sheet; C, coil; T, turn; -, no prediction. QF cleavagesite is indicated in bold.^(b)Garnier J. et al. (1987) J. Mol. Biol. 120:97-120.^(c)McCleland D. G, Rumelhart D. E. In Explorations in ParallelDistributed Processing. 3:318-362. 1988. MIT Press, Cambridge MA;Kneller D. G., et al. (1990) J. Mol. Biol. 214:171-182.

These Previns or core structures are generally described by the formula:B₁Z*₂B₃Z*₄X*₅Q₆F₇X₈X₉X₁₀X₁₁   (1),B₁X₂X₃X₄X₅Q₆F₇X₈X₉X₁₀X₁₁   (2), orB₁Z₂B₃X₄Z₅Q₆F₇Z₈X₉X₁₀X₁₁   (3)and the salts, esters, amides, and acyl forms thereof. Each positionrepresented by a letter indicates a single amino acid residue: B is abasic or polar/large amino acid or a modified form thereof, X is a smallor hydrophobic amino acid or a modified form thereof, X* is a small orpolar/large amino acid or a modified form thereof, Z is a polar/large orhydrophobic amino acid or a modified form thereof; Z* is Proline or apolar/large or hydrophobic amino acid or a modified form thereof. Asdescribed below, one or more of the peptide linkages between the aminoacid residues may be replaced by a peptide linkage mimic.

The invention compounds include those core structures represented byformula (1), (2) and (3).

The amino terminus of the peptide may be in the free amino form or maybe acylated by a group of the formula RCO—, wherein R represents ahydrocarbyl group of 1-6C. The hydrocarbyl group is saturated orunsaturated and is typically, for example, methyl, ethyl, i-propyl,t-butyl, n-pentyl, cyclohexyl, cyclohexene-2-yl, hexene-3-yl,hexyne-4-yl, and the like.

The C-terminus of the peptides of the invention may be in the form ofthe underivatized carboxyl group, either as the free acid or anacceptable salt, such as the potassium, sodium, calcium, magnesium, orother salt of an inorganic ion or of an organic ion such as caffeine.The carboxyl terminus may also be derivatized by formation of an esterwith an alcohol of the formula ROH, or may be amidated by an amine ofthe formula NH₃, or RNH₂, or R₂NH, wherein each R is independentlyhydrocarbyl of 1-6C as defined above. Amidated forms of the peptideswherein the C-terminus has the formula CONH₂ are preferred.

The peptides of the invention may be supplied in the form of the acidaddition salts. Typical acid addition salts include those of inorganicions such as chloride, bromide, iodide, fluoride or the like, sulfate,nitrate, or phosphate, or may be salts of organic anions such asacetate, formate, benzoate and the like. The acceptability of each ofsuch salts is dependent on the intended use, as is commonly understood.

The amino acids in the peptides of the invention may be those encoded bythe gene or analogs thereof, and may also be the D-isomers thereof. Apreferred embodiment is a compound having the core structure of theformula (1), (2) or (3) wherein the compound is resistant to proteaseactivity by having at least some of its residues in the D-configurationand retains the ability to inhibit BttxB protease activity.

The amino acid notations used herein are conventional and are asfollows: Amino Acid One-Letter Symbol Three-Letter Symbol Alanine A AlaArginine R Arg Asparagine N Asn Aspartic acid D Asp Cysteine C CysGlutamine Q Gln Glutamic acid E Glu Glycine G Gly Histidine H HisIsoleucine I Ile Leucine L Leu Lysine K Lys Methionine M MetPhenylalanine F Phe Proline P Pro Serine S Ser Threonine T ThrTryptophan W Trp Tyrosine Y Tyr Valine V Val

The compounds of the invention are peptides or peptide-like compoundswhich are partially defined in terms of amino acid residues ofdesignated classes. Amino acid residues can be generally subclassifiedinto major subclasses as follows:

Acidic: The residue has a negative charge due to loss of H ion atphysiological pH and the residue is attracted by aqueous solution so asto seek the surface positions in the conformation of a peptide in whichit is contained when the peptide is in aqueous medium at physiologicalpH.

Basic: The residue has a positive charge due to association with H ionat physiological pH or within one or two pH units thereof (e.g.,histidine) and the residue is attracted by aqueous solution so as toseek the surface positions in the conformation of a peptide in which itis contained when the peptide is in aqueous medium at physiological pH.

Hydrophobic: The residues are not charged at physiological pH and theresidue is repelled by aqueous solution so as to seek the innerpositions in the conformation of a peptide in which it is contained whenthe peptide is in aqueous medium.

Neutral/polar: The residues are not charged at physiological pH, but theresidue is not sufficiently repelled by aqueous solutions so that itwould seek inner positions in the conformation of a peptide in which itis contained when the peptide is in aqueous medium. This descriptionalso characterizes certain amino acids as “small” since their sidechains are not sufficiently large, even if polar groups are lacking, toconfer hydrophobicity. “Small” amino acids are those with four carbonsor less when at least one polar group is on the side chain and threecarbons or less when not.

It is understood, of course, that in a statistical collection ofindividual residue molecules some molecules will be charged, and somenot, and there will be an attraction for or repulsion from an aqueousmedium to a greater or lesser extent. To fit the definition of“charged,” a significant percentage (at least approximately 25%) of theindividual molecules are charged at the relevant pH. The degree ofattraction or repulsion required for classification as polar or nonpolaris arbitrary and, therefore, amino acids specifically contemplated bythe invention have been classified as one or the other. Most amino acidsnot specifically named can be classified on the basis of known behavior.

Amino acid residues can be further subclassified as cyclic or noncyclic,and aromatic or nonaromatic, self-explanatory classifications withrespect to the side-chain substituent groups of the residues, and assmall or large. The residue is considered small if it contains a totalof four carbon atoms or less, inclusive of the carboxyl carbon, providedan additional polar substituent is present; three or less if not. Smallresidues are, of course, always nonaromatic.

For the naturally occurring protein amino acids, subclassificationaccording to the foregoing scheme is as follows. Acidic Aspartic acidand Glutamic acid Basic Noncyclic: Arginine, Lysine Cyclic: HistidineSmall Glycine, Serine, Alanine, Threonine, Cysteine Polar/largeAsparagine, Glutamine Hydrophobic Tyrosine, Valine, Isoleucme, Leucine,Methionine, Phenylalanine, Tryptophan

The gene-encoded secondary amino acid proline is a special case due toits known effects on the secondary conformation of peptide chains, i.e.helix structure disruptions. Therefore, proline may only be allowed inposition 26 where it would help to disrupt the helix structures found onboth sides of the QF cleavage site and force the helix-turn-helixstructure.

Cysteine is a small amino acid. Generally, there are no cysteine ormethionine in the sequences of the VAMP2 substrate, B-I, B-II, Peptide24, Peptide 36. The side chain of cysteine is somewhat hydrophobic, butit is highly reactive. The sulfur moiety has the potential to react withthe sulfur in other cysteine to from a cystine or disulfide bond.Cysteine may be modified so as to prevent its participation in thesecondary structure. Additionally, cysteine may be used as a spaceranchor in a compound of formula (1), (2) or (3). Furthermore, it may beadvantageous to incorporate cysteine for use as a reactive site to labela core structure with fluorescent markers.

The “modified” amino acids that may be included in the core structuresare gene-encoded amino acids which have been processed after translationof the gene, e.g., by the addition of methyl groups or derivatizationthrough covalent linkage to other substituents or oxidation or reductionor other covalent modification. The classification into which theresulting modified amino acid falls will be determined by thecharacteristics of the modified form. For example, if lysine weremodified by acylating the, -amino group, the modified form would not beclassed as basic but as polar/large amino acid.

Certain commonly encountered amino acids, which are not encoded by thegenetic code, include, for example, beta-alanine (beta-Ala), or otheromega-amino acids, such as 3-aminopropionic, 2,3-diaminopropionic(2,3-diaP), 4-aminobutyric and so forth, alphaaminisobutyric acid (Aib),sarcosine (Sar), ornithine (Om), citrulline (Cit), t-butylalanine(t-BuA), t-butylglycine (t-BuG), N-methylisoleucine (N-McIle),phenylglycine (Phg), and cyclohexylalanine (Cha), norleucine (Nle),2-naphthylalanine (2-Nal); 1,2,3,4-tetrahydroisoquinoline-3-carboxylicacid (Tic); ∃-2-thienylalanine (Thi); methionine sulfoxide (MSO); andhomoarginine (Har). These also fall conveniently into particularcategories.

-   -   Based on the above definitions,    -   Sar, beta-Ala and Aib are small;    -   t-BuA, t-BuG, N-McIle, Nle, Mvl, Cha, Phg, Nal, Thi and Tic are        hydrophobic;    -   2,3-diaP, Orn and Har are basic;    -   Cit, Acetyl Lys and MSO are neutral/polar/large.

The various omega-amino acids are classified according to size as small(beta-Ala and 3-aminopropionic) or as large and hydrophobic (allothers).

Other amino acid substitutions, which are not gene encoded, are includedin peptide compounds within the scope of the invention and can beclassified within this general scheme according to their structure. Forexample, D-amino acid substitutions would be desirable to circumventpotential stability problems due to endogenous protease activity;especially important for an oral dosage route.

In all of the compounds of the invention, one or more amide linkages(—CO—NH—) may optionally be replaced with another linkage which is anisostere such as —CH₂NH—, —CH₂S—, —CH₂CH₂, —CH═CH— (cis and trans),—COCH₂—, —CH(OH)CH₂— and —CH₂SO—. This replacement can be made bymethods known in the art. The following references describe preparationof peptide analogs which include these alternative-linking moieties:Spatola, A. F., Vega Data (March 1983), Vol. 1, Issue 3, “PeptideBackbone Modifications” (general review); Spatola, A. F., in “Chemistryand Biochemistry of Amino Acids Peptides and Proteins,” B. Weinstein,eds., Marcel Dekker, New York, p. 267 (1983) (general review); Morley,J. S., Trends Pharm Sci (1980) pp. 463-468 (general review); Hudson, D.,et al., Int J Pept Prot Res (1979) 14:177-185 (—CH₂NH—, —CH₂CH₂—);Spatola, A. F., et al., Life Sci (1986) 38:1243-1249 (—CH2-S); Hann, M.M., J Chem Soc Perkin Trans I (1982) 307-314 (—CH—CH—, cis and trans);Almquist, R. G., et al., J Med Chem (1980) 23:1392-1398 (—COCH₂—);Jennings-White, C., et al., Tetrahedron Lett (1982) 23:2533 (—COCH₂—);Szelke, M., et al., European Application EP 45665 (1982) CA:97:39405(1982) (—CH(OH)CH₂—); Holladay, M. W., et al., Tetrahedron Lett (1983)24:4401-4404 (—C(OH)CH₂—); and Hruby, V. J., Life Sci (1982) 31:189-199(—CH₂—S—).

Typical compounds within the scope of the invention are:    RPKPQQFFGLM(SEQ ID NO:1) TRSSRAGLQFPVGRVHRLLRK (SEQ ID NO:2) TRAARAGLQFPVGRVHRLLRK(SEQ ID NO:3) TRLLRAGLQFPVGRVHRLLRK (SEQ ID NO:4)    RAKPQQFFGLM (SEQ IDNO:5)    RAKAQQFFGLM (SEQ ID NO:6)    RAKAQQFPGLM (SEQ ID NO:7)   RAKLQQFPGLM (SEQ ID NO:8)    RAKGLQFPGLM (SEQ ID NO:9)    RAGLGQFFGLM(SEQ ID NO:10) DAARAKGLQFPGLMAKLK (SEQ ID NO:11) DAARAKGLQFPGLLAKLK (SEQID NO:12) TRSRAKGLQFPGLMVHRL (SEQ ID NO:13)       XQF------------------------Y (SEQ ID NO:14)wherein “-” is a spacer molecule and “X” is any suitable amino acidsequence homologous to those upstream of the QF site of SEQ ID NOs: 1-12

“Active” compounds are defined as those compounds having the coresequence of the invention and inhibit BttxB and/or Tttx proteaseactivities. The conformation of the compounds of the invention may bedetermined by circular dichroism. See C B naves, J. M., et al. (1998) J.Biol. Chem. 273:43214-34221. Proton NMR may also be used. See Yi, G. etal. (1996) FEBS Lett. 398:87-90. X-ray crystallography may also be used.See Sutton, R. B., et al. (1998) Nature 395, 347-353.

“Derivatives” are defined as those compounds having a core sequence ofthe invention and contain amino acid modifications comprising‘unnatural’ amino acids other than the known 21 amino acids (20 common,and then selenocysteine, which is an uncommon but naturally occurringnon-gene encoded amino acid) or additions such as cysteine and lysine ontermini to provide a reactive center for conjugation to other chemicals,labels or proteins.

Compounds of the formula (1), (2) or (3) may be used to constructcompounds optimized for inhibiting the protease activity of BttxB orTttx. Optimization may be done by substituting amino acids that promotehelical structure formation upstream of the QF site. Examples of suchare peptides having the amino acid sequences are shown in FIG. 7.

Substance P is a weak inhibitor of BttxB. It contains an alpha helicalstructure downstream of a QF site. It does not have an alpha helicalstructure upstream of the QF site. Computer modeling has been used toconstruct compounds which are similar to Substance P, yet contain alphahelices upstream and downstream of the QF site. Thus, compounds such asSubstance P may be utilized as a molecular building block forconstructing more potent BttxB inhibitors which potent inhibitors maycomprise Buforinins. Therefore Substance P is considered a corestructure within the scope of the compounds of the invention.

For example, Substance P may be modified by 4 substitutions, P2A, G3K,P4L, Q5G, to obtain a compound having the sequence, RAGLGQFFGLM. Thesesubstitutions are predicted to generate the helical structures requiredfor inhibition of BttxB protease activity. See Garcia et al. J. AppliedToxicology, in press.

In another embodiment, a spacer could be utilized in a Substance P likemolecular building block in order to place a Tyrosine (˜21 angstroms)from the F residue of the QF site as the location of Tyr is importantsince its removal in the B-I should a decrease in inhibition.

Suitable spacers include: Bis-Maleimidethane (BMOE) 8 Angstroms spacerarm; 1,4-Bis-maleimidobutane (BMB) 10.9 Angstoms spacer arm. See Chen,L. L. et al. J. Biol. Chem. 266:18237-18243, Yi, F. et al. J. Biol.Chem. 266:3900-3906.

As shown below, there is an apparent similarity in the position of Tyrfor B-I, VAMP2 and peptide 36: AGRGKQGGKVRAKAKTRSSRAGLQFPVGRVHRLLRKGNYBuforin I   ERDQKLSELDDRADALQAGASQFETSAAKLKRKYWWKNLK VAMP2₅₄₋₉₄AGRGKQGGKVRAKAKTRSSRAGLQFPVGRVHRLLRK peptide36              RPKPQQFFGLM-------YYY

The deletion of the C-terminal three amino acids results in a decreaseof inhibition of BttxB by 50% as compared with B-I. See Garcia et al.(1999) J Applied Toxicol. in press. This indicates that the position ofthe Tyr residue may be important for the inhibitory function of thecompounds.

Therefore a compound may be created from a Substance P like molecularbuilding block to enhance its BttxB inhibitory ability by modifying theplacement of Tyr or other hydrophobic amino acids an optimal distancefrom the QF cleavage site. This distance could be provided by peptidesequence or other suitable spacer molecules.

For example, a spacer incorporated upstream from the QF site in V2sequence which would place Tyr around 6 angstroms from the Q of the QFsite could improve inhibition. See Whitcome et al. (1996) FEBS Let.386:133-136 (ELDDRADALQ).

Spacers may be used to cross-link individual components to reach thedesired distance. Such spacers include carbon chains of desiredrepetitions and length. A carbon chain spacer would be advantageous asit would confer resistance to cleavage and for esterification andamidation as well as neutralizing in the presence of base to form saltsare all standard organic chemical techniques.

If the core structures of the invention are prepared under physiologicalconditions, the side-chain amino groups of the basic amino acids will bein the form of the relevant acid addition salts.

If the core structure is a peptide backbone comprised entirely ofgene-encoded amino acids, or if some portion of it is so composed, thepeptide or the relevant portion may also be synthesized usingrecombinant DNA techniques. The DNA encoding the core structures of theinvention may be synthesized using standard techniques in the art suchas solid phase DNA synthesis with conventional equipment that includes,for example, an ABI 3948 Nucleic Acid Synthesis System (Perkin ElmerApplied Biosystems, Foster City, Calif.) utilizing phosphoramiditesynthesis chemistry (Beaucage, S. L. et al. (1981) Tetrahedron Lett.22:1859-1862). DNA oligomers would be synthesized with overlappingmatching complimentary sequences. Annealing of these sequences wouldform a double-stranded synthetic gene. Building on this process wouldgive larger and larger double-stranded products till the requisite geneis built. Alternatively, DNA recombinant means would be employed bycloning the core structure of the invention, compounds containing thecore structure, or like-fragment of H2A protein, and then modifying bysite-directed mutagenesis or DNA-cassette replacement or other means inthe art (Methods Enzymology vol. 152; Eds. S. L. Berge and A. R. Kimmel,Academic Press, Inc., Orlando, Fla., 1998) to achieve the modificationdesired. Codon choice can be integrated into the synthesis depending onthe nature of the host.

For recombinant production, the DNA encoding the core structure isincluded in an expression system which places these coding sequencesunder the control of a suitable promoter and other control sequenceswhich are compatible with an intended host cell. Types of host cellsavailable span almost the entire range of the plant and animal kingdoms.Thus, the core structures of the invention or compounds containing thecore structures could be produced in bacteria or yeast (to the extentthat they can be produced in a nontoxic or refractile form or utilizeresistant strains) as well as in animal cells, insect cells and plantcells.

The core structures of the invention or compounds containing the corestructures can be produced in a form that will result in their secretionfrom the host cell by fusing to the DNA encoding the core structure ofcompound containing the core structure, a DNA encoding a suitable signalpeptide, or may be produced intracellularly. They may also bedegradation. Spacers and use of spacers are known in the art. See e.g.,Synthetic Peptides Ed. G. A. Gant, W. H. Freeman & Co. New York, N.Y.,1992.

Alternatively, one may use amino acid residues to place the activemoieties at appropriate distances from the QF site or create enhancingstructures. For example, Cys may be used to manipulate a compound tohave or not have a particular structure such as a disulfide loop.However, the use of other compounds such as TCEP, may adversely interactwith such manipulated structures, i.e. the disulfide loop would beopened.

Consequently, one may use a Previn or a core structure of the formula(1), (2) or (3) to construct a Buforinin like compound or anothercompound which highly inhibits the protease activity of BttxB.

Preparation of the Invention Compounds

The invention compounds, often designated herein “Previns” areessentially core structures or molecular building blocks which may bemodified at the N- or C-terminus and optimized for constructingcompounds which inhibit the protease activity of BttxB or Tttx.

Standard methods can be used to synthesize core structures similar insize and conformation to the Previns. Most commonly used currently aresolid phase synthesis techniques; indeed, automated equipment forsystematically constructing peptide chains can be purchased. Solutionphase synthesis can also be used but is considerably less convenient.When synthesized using these standard techniques, amino acids notencoded by the gene and D-enantiomers can be employed in the synthesis.

The N- and/or C-terminus can be modified with conventional chemicaltechniques. The compounds of the invention may optionally contain anacyl or an acetyl group at the amino terminus. Methods for acetylatingor, more generally, acylating, the free amino group at the N-terminusare generally known in the art.

At the carboxy terminus, the carboxyl group may be present in the formof a salt; and in the case of pharmaceutical compositions, the salt willbe a pharmaceutically acceptable salt. Suitable salts include thoseformed with inorganic ions such as NH₄ ⁺, Na⁺, K⁺, Mg⁺⁺, Ca⁺⁺, and thelike as well as salts formed with organic cations such as those ofcaffeine and other highly substituted amines. The carboxy terminus mayalso be esterified using alcohols of the formula ROH wherein R ishydrocarbyl (1-6C) as defined above. Similarly, the carboxy terminus maybe amidated so as to have the formula —CONH₂, —CONHR, or —CONR₂, whereineach R is independently hydrocarbyl (1-6C) as herein defined. Techniquesproduced as fusion proteins with additional amino acid sequence whichmay or may not need to be subsequently removed prior to the use of thesecompounds as an inhibitor of BttxB protease activity.

Thus, the core structures of the invention can be produced in a varietyof modalities including chemical synthesis and recombinant production orsome combination of these techniques.

Any members of the Previn class which occur naturally are supplied inpurified and isolated form. By “purified and isolated” is meant freefrom the environment in which the peptide normally occurs (in the caseof such naturally occurring peptides) and in a form where it can be usedpractically. Thus, “purified and isolated” form means that the peptideis substantially pure, i.e., more than 90% pure, preferably more than93% pure and more preferably more than 99% pure or is in a completelydifferent context such as that of a pharmaceutical preparation.

The invention is also directed to the screening assays for compoundscontaining the core structures and assays utilizing the core structuresand compounds containing the core structures.

The invention is also directed to the use of compounds containing thecore structures as intracellular inhibitors of BttxB. Bttxs specificallytarget nerve cells because of the receptor-like recognition of cellsurface gangliosides and synaptogamin by the nerve-cell targeting heavychain (HC) subunit of the toxin. See Kozaki, S., et al. (1998) Microb.Pathog. 25:91-99. Once bound, the toxin is internalized by a mechanismnot completely understood but apparently requires acidification of theendosome and cleavage of the disulfide bond linking the HC and theendoproteolytically active light chain (LC).

The specificity of this delivery system would be useful for delivery ofcompounds containing the core structures to those cell types poisoned orpotentially poisoned with BttxB and could be used as a ‘magic bullet’since the magic bullet approach is becoming a reality. See e.g. Pastan,L, et al. (1994) Ann. Rev. Biochem. 61:331-354 and Engert, A., et al.(1998) Curr. Top. Microbial. Immunol. 234:13-33 (Introduction ofimmunotoxins linked to Diptheria toxin or Ricin A chain).

Therefore, the core structures or compounds containing the corestructures may be linked to BttxB HC with a linkage such as a disulfidebond. Alternatively, the core structures or compounds containing thecore structures may be linked to BttxB HC with a carrier protein such ashuman albumin or another bridge to form a multi-protein conjugate. Thisconjugate should then target the susceptible cells in a manner similarto BttxB. Once inside the cell, the conjugate may inhibit BttxB or thelinkage may be cleaved to free the compound containing the corestructure or carrier-core structure to inhibit BttxB protease activity.

Antibodies

Antibodies to the core structures may be produced using standardimmunological techniques for production of polyclonal antisera and, ifdesired, immortalizing the antibody-producing cells of the immunizedhost for sources of monoclonal antibody production. Techniques forproducing antibodies to any substance of interest are well known. It maybe necessary to enhance the immunogenicity of the substance,particularly as here, where the material is only a short peptide, bycoupling the hapten to a carrier. Suitable carriers for this purposeinclude substances which do not themselves produce an immune response inthe mammal to be administered the hapten-carrier conjugate. Commoncarriers used include keyhole limpet hemocyanin (KLH), diphtheriatoxoid, serum albumin, and the viral coat protein of rotavirus, VP6.Coupling of the hapten to the carrier is effected by standard techniquessuch as contacting the carrier with the peptide in the presence of adehydrating agent such as dicyclohexylcarbodiimide or through the use oflinkers such as those available through Pierce Chemical Company,Chicago, Ill.

The core structures or compounds containing the core structures inimmunogenic form are then injected into a suitable mammalian host andantibody titers in the serum are monitored.

Polyclonal antisera may be harvested when titers are sufficiently high.Alternatively, antibody-producing cells of the host such as spleen cellsor peripheral blood lymphocytes may be harvested and immortalized. Theimmortalized cells are then cloned as individual colonies and screenedfor the production of the desired monoclonal antibodies. The genesencoding monoclonal antibodies secreted by selected hybridomas or othercells may be recovered, manipulated if desired, for example, to providemultiple epitope specificity or to encode a single-chain form and may beengineered for expression in alternative host cells, such as CHO cells.

Thus, as used herein, “antibodies” also includes any immunologicallyreactive fragment of the immunoglobulins such as Fab, Fab′ and F(ab′)₂fragments as well as modified immunoreactive forms such as Fv regions,which are produced by manipulation of the relevant genes (isolable, forexample, from the appropriate hybridoma).

The antibodies of the invention are, of course, useful in immunoassaysfor determining the amount or presence of the core structures. Suchassays are essential in quality controlled production of compositionscontaining the core structures of the invention. In addition, theantibodies can be used to assess the efficacy of recombinant productionof the core structures, as well as for screening expression librariesfor the presence of Previn encoding genes. They may also be used asaffinity ligands for purifying and/or isolating the core structures andcompounds containing the core structures. They may also be used fordetecting and measuring core structures in sera or plasma by methodswell known in the art such as RIA and ELISA. Therefore, one may monitorcirculating Previns or compounds containing the core structures of theinvention levels to assure sufficient dosage.

Compositions Containing the Previns and Methods of Use

The core structures are useful in constructing compounds that areeffective in inhibiting the protease activity of bttxB and tetanusneurotoxins. Accordingly, compounds containing the core structures ofthe invention can be used in prevention, prophylaxis and therapies forBttxB and Tttx poisoning. For use in such contexts, a compoundcontaining the core structure may be administered alone, or a variety ofcompounds containing the core structure and free Previns may beadministered. Furthermore, additional protease inhibitors or adjunctchemicals such as tris-(2-carboxyethl)phosphine (TCEP) may beadministered along with Previns or compounds containing the corestructures of the invention.

TCEP is a non-odorous, non-sulfhydryl containing reducing agent that isrelatively non-toxic in animals (P—CH₂CH₂COOH)₃HCl; Molecular Probes,Inc. Eugene Oreg.). TCEP can reduce the disulfide bond between the HCand LC and allow the dissociation of the BttxB or Tttx subunits. Thisdissociation increases the availability of the active QF site tocompounds which inhibit BttxB protease activity. Additionally, thedisassociation of the toxin prevents nerve cell penetration. Otherreducing agents such as dithiothreitol (DTT) may be used; however, theymay be objectionable due to their distinctive odors and toxicity.Therefore, TCEP is preferred.

The core structures of the invention are also useful as standards inmonitoring assays and in assays for evaluating the effectiveness oflater-generation compounds containing the core structures. This could bedone by utilizing the endopeptidase activity assay for BttxB. In thisendopeptidase assay, one may evaluate whether potential peptidesfunction as inhibitors or substrates of BttxB by the ability to cleaveof a synthetic peptide substrate comprising amino acids 55-94 of theintracellular target VAMP2. The cleavage products may be separated by aC₁₈ reverse-phase HPLC column and detected by absorbance at 205 nm.

For preventing the initial intoxication or further poisoning caused byBttxB and Tttx in animal subjects, the core structures or compoundscontaining the core structures can be formulated as pharmaceutical orveterinary compositions. Depending on the subject to be treated, themode of administration, and the type of treatment desired—e.g.,prevention, prophylaxis, therapy; the core structures and the compoundscontaining the core structures are formulated in ways consonant withthese parameters. A summary of such techniques is found in Remington'sPharmaceutical Sciences, latest edition, Mack Publishing Co., Easton,Pa.

In general, for use in treatment or prophylaxis, the Previns andcompounds containing the core structure may be used alone or incombination with other compounds which inhibit protease activity such asVAMP2. Use of the enantiomeric forms containing all D-amino acids mayconfer advantages such as resistance to those proteases, such as trypsinand chymotrypsin.

The Previns and compounds containing thecore structures can beadministered singly or as mixtures of several Previns and compounds orin combination with other pharmaceutically active components, and insingle or multiple administrations. The formulations may be prepared ina manner suitable for systemic administration. Systemic formulationsinclude those designed for injection, e.g. intramuscular, intravenous orsubcutaneous injection, or may be prepared for transdermal,transmucosal, or oral administration. The formulation will generallyinclude a diluent as well as, in some cases, adjuvants, buffers,preservatives and the like. The Buforinins can be administered also inliposomal compositions or as microemulsions using conventionaltechniques.

If orally administered, the compounds of the invention must be protectedfrom degradation in the stomach using a suitable enteric coating. Thismay be avoided to some extent by utilizing amino acids in theD-configuration, thus providing resistance to protease. However, thepeptide is still susceptible to acid hydrolysis; thus, some degree ofenteric coating may still be required.

The manner of administration and formulation of the compounds useful inthe invention and their related compounds will depend on the nature ofthe condition, the severity of the condition, the particular subject tobe treated, and the judgment of the practitioner; formulation willdepend on mode of administration. As the compounds of the invention aresmall molecules, they are conveniently administered by oraladministration by compounding them with suitable pharmaceuticalexcipients so as to provide tablets, capsules, syrups, and the like.Suitable formulations for oral administration may also include minorcomponents such as buffers, flavoring agents and the like. Typically,the amount of active ingredient in the formulations will be in the rangeof 5%-95% of the total formulation, but wide variation is permitteddepending on the carrier. Suitable carriers include sucrose, pectin,magnesium stearate, lactose, peanut oil, olive oil, water, and the like.

The compounds useful in the invention may also be administered throughsuppositories or other transmucosal vehicles. Typically, suchformulations will include excipients that facilitate the passage of thecompound through the mucosa such as pharmaceutically acceptabledetergents.

The compounds may also be administered topically, for topical conditionssuch as psoriasis, or in formulation intended to penetrate the skin.These include lotions, creams, ointment and the like which can beformulated by known methods.

The compounds may also be administered by injection, includingintravenous, intramuscular, subcutaneous or intraperitoneal injection.Typical formulations for such use are liquid formulations in isotonicvehicles such as Hank's solution or Ringer's solution.

Suitable alternative formulations also include nasal sprays, liposomalformulations, slow-release formulations, and the like.

Any suitable formulation may be used. A compendium of art-knownformulations is found in Remington's Pharmaceutical Sciences, latestedition, Mack Publishing Company, Easton, Pa. Reference to this manualis routine in the art.

A preferred means to deliver the Previns and compounds containing thecore structures of the invention would include the use TCEP. Since TCEPcleaves the holotoxin which yields a site available to the activemoieties of the core structures. TCEP also disassociates the toxins intoindividual components which prevents nerve cell penetration. Also, thecore structures could be coupled to a variety of compounds including aBttxB heavy chain, which excludes the toxin light chain, to target thePrevin or the compound containing the core structure to the toxinaffected cells.

The dosages of the compounds of the invention will depend on a number offactors which will vary from patient to patient. The following examplesare intended to illustrate but not to limit the invention.

EXAMPLE 1 Endopeptidase Activity Assay

The toxin was activated immediately prior to use by incubating at 25° C.for 30 minutes in an activation mixture that contained, in a volume of7.5 μl per digest: 2.4 μg (16 pmol) of toxin, 30 mM NaHEPES buffer, pH7.3, and 5 mM DTT or TCEP. A substrate peptide mix was prepared thatcontained 1 nmol of the substrate peptide (VAMP2 55-94), 4% DMSO, 4%Triton X-100, and 80 mM NaHEPES buffer, pH 7.3, per digest. The finalreaction mix was made by adding 25 μl of the substrate peptide mix, 4.5μl of fresh 10 mM DTT, 13 μl H₂O or test peptide, and 7.5 μl ofactivation mixture. The reaction was initiated by incubation at 37° C..The reaction was stopped by the addition of 1 vol trifluoroacetic acid(TFA) to 0.25%. The samples were clarified by centrifligation.

In this assay, 16 pmol of BttxB digested 1 nmol of the substrate tocompletion in less than 45 min. at 37° C..

EXAMPLE 2 Reverse Phase HPLC Analysis of Digestion Products

Digested peptide products were fractionated by RP-HPLC on aWaters:Bondapak analytical C₁₈ column (3.9 mm×30 cm) attached to Beckman126 pumps and a model 168 Diode Array Detector, controlled by BeckmanSystem Gold Ver 8.1 software. The solvent system consisted of buffer A(BA; H₂O-0.1% TFA) and buffer B (BB; CH₃CN-0.1%TFA). The developmentprogram consisted of the following: 97% BA, 0-1 min; to 33% BB, 1-30min; then wash with 97% BB for 5 min, followed by equilibration in 97%BA for 10 min. The flow rate was ml min⁻¹ except during the wash andequilibrium phase where it was 1.5 ml min⁻¹. 75 μl injections were madewith a Waters Intelligent Sample Processor (WISP Model 712). Theeffluent was monitored at dual wavelengths of 205 and 280 nm.

Initially, digestion products are identified by peptide sequencing usingautomated Edman-degradation on an ABI 477A protein sequencer attachedin-line with a HPLC (ABI model 120A) for detection ofphenlythiohydratoin derivatized amino acids. The extent of digestion wasdetermined by comparison of peak areas of undigested controls (no addedtoxin) and total digests (digests allowed to go to completion, typically2-3 h). The extent of inhibition or digestion will be determined fromexamination of the chromatograms by peak area comparison with standardsand/or products formed compared with quantified standards or digestswithout added inhibitor that have gone to completion.

EXAMPLE 3 Secondary Structure Predictions

Secondary structures were predicted by using the nnpredict, and theGibrat (GOR2) programs. See McCleland, D. G, Rumelhart D. E. InExplorations in Parallel Distributed Processing. vol. 3:318-362. 1988.MIT Press, Cambridge Mass.; Kneller D. G., et al. (1990) J. Mol. Biol.214:171-182; Garnier, J. et al. (1978) J. Mol. Biol 198;425-443; GarnierJ. et al. (1987) J. Mol. Biol. 120:97-120; Garnier, J., et al. (1996)Methods Enzymol. 266:540-553. Helical wheel projections were made usingthe Antheprot program Ver 4. See Deleage, G., Instit de Biologie etChimi des Proteins, Lyon, France.

The Gibrat program predicts that B-I could form analpha-helical-turn-alpha-helical configuration similar to that of VAMP2.See Table 3. The result that Buforin I may form a secondary structuresimilar to VAMP2 then suggests that B-I may also form a similarsupersecondary structure of a reverse turn with helix bundling similarto VAMP2. See Lebeda, et. al. (1996). In support of this prediction, wefound that the diminishing inhibition of BttxB activity and its helicalcontent as Buforin-I was truncated, mirrors the diminishing activity ofBttxB for substrate deletions. See data for Buforin-II in Table 1 andFIG. 4.

EXAMPLE 4 Preparation of Buforinins, Previns and Compounds Containingthe Core Structures

The Buforinins and compounds containing the core structures may beobtained from amphibian stomach by gut lavage using methods as describedby Park, C. B. et al. See Park, C. B., et al. (1996) Biochem. Biophys.Res. Comm. 218:408-413.

The Buforinins and compounds containing the core structures may besynthesized by solid-phase peptide synthesis (SSPS) as described by L.A. Carpino, J. Am. Chem. Soc. 79,4427 (1957), C. D. Chang et al., Int.J. Pept. Protein Res. 11,246 (1978), E. Atherton, et al., J. Chem Soc.Chem. Commun., 537 (1978) and R. B. Merrifield, J. Am. Chem. Soc. 85,2149 (1963) and Barlos, K., et al., (1989) Tetrahedron Lett. 30:3947.

The Buforinins, Previns and compounds containing the core structures mayalso be produced by DNA recombinant means commonly known in the artwhereby a suitable promoter for expression in heterologous systems, i.e.bacterial, fungi, insect, or mammalian cell cultures may be used. TheDNA sequence may be optimized for the particular host and tRNA content.For example, a compound such as a Buforinin or other which contains thecore sequence of the invention may be enzymatically digested to isolatethe core sequence.

Once isolated, recombinant means may be used to modify the core sequenceor add additional amino acids or other moieties onto the core sequencein order to make a compound that effectively inhibits the proteaseactivity of BttxB or Tttx.

EXAMPLE 5 Inhibition of Protease Activity by Buforinins

The endopeptidase assay and reverse phase HPLC as described in Examples1 and 2 may be used to detect the cleavage products and the extent ofprotease inhibition. Briefly, potential inhibitors may be added to thesubstrate peptide mix immediately before the addition of the activationmix containing the toxin as described in Garica, et al. After incubationfor 45 min at 37° C., the reaction should be stopped and the digestionproducts may be analyzed by using RP HPLC. If a fluorescent-labeledsubstrate is used then product formation will be determined with anin-line fluorescent detector.

The extent of inhibition or digestion will be determined as described inExample 2 of undigested substrate remaining and/or products formedcompared with quantified standards or digests without added inhibitorthat have gone to completion.

Alternative means can be used include densitometry wherein thesubstrates and products separated by electrophoresis and stained withprotein specific dyes, i.e. Coomassie brilliant blue, and measured. Onemay also perform immunoassays to determine the extent of inhibition ordigestion by utilizing substrate or product specific antibodies.

Alternatives also include in vivo protection or tissue-specific functionassays. For example, an experimental animal would be dosed with theinhibitor with or with out adjuncts and then challenged with the toxin,e.g. i.v. injection of a Buforinin with a reducing agent such as TCEP.The onset of symptoms or an alteration of the LD₅₀ would then beevaluated. Tissue protection assays would employ an intactnerve-muscle-preparation wherein muscle twitch response to nerve cellstimulation would be evaluated. The toxin would be preincubated with aBuforinin and adjuncts and are then added to the tissue preparation.

EXAMPLE 6 Designing Buforinins, Previns and Other Compounds having theCore Sequences of the Invention with an Effect on BttxB ProteaseActivity

By using standard methods and techniques, the peptides of the inventionmay be modified by either making mutations or substitutions whichinclude substituting Pro₂₆ with glutamine to make the active site morelike the substrate, or other amino acid, that favors turn formationwithout the turn constraint imposed by Pro. Such substitutions arepredicted to result in more effective helix bundling for toxinassociation to occur. Other amino acid substitutions or mutations in thehelix region could be made so that either the helix becomes moreamphipathic to improve helix bundling or improve interaction with thetoxin. Such changes would include a substitution of R11 with L oranother helix favoring amino acid. See FIGS. 6A and B. Similarly,multiple substitutions R11L, K15L, and S18L or other amino acids couldbe made to favor helix formation and bundling.

Alternatively, B-II which lacks the predicted upstream helix of B-I maybe modified to enhance and improve its ability to inhibit BttxB proteaseactivity. For example, a peptide having substitutions S3A and S4A (SEQID NO:5) has a predicted helix upstream of the QF site. Another examplewould be a peptide having substitutions S2L and S4L (SEQ ID NO:6).Likewise, this peptide has a predicted helix upstream of the QF site.

EXAMPLE 7 Pretreatment with Buforinins, Previns and Other Compoundshaving a Core Sequence of the Invention

Buforinins, Previns, and other compounds having the core sequence of theinvention may be used to pretreat food and liquids that might becontaminated with BttxB or Tttx. For example, an effective amount of aBuforinin, Previn, or other compound having the core sequence may bemixed into water having BttxB to inhibit the protease activity of theBttxB, e.g. 100 ml of water containing 1 ug of BttxB would be treatedwith 100 ug of a Buforinin, Previn, or other compound and 0.1 mmmolreducing agent, i.e. TCEP in tablet, powder, or liquid form.

These various forms would comprise of a Buforinin, Previn, or othercompound having the core structure, reducing agent such as TCEP, andother fillers and stabilizers. A liquid form could be made from a tabletor powder that is pre-dissolved prior to use. A solution having aBuforinin, Previn, or other compound containing a core structure of theinvention may be applied on the surface of solid food having BttxB onthe surface. Alternatively, an effective amount of the solution may beused to treat solid food which has been ground into small particles inorder to allow the active ingredient of the solution access to amountsof BttxB which is not found on the surface of the food.

Contaminated or suspect non-food surfaces may also be washed withsolutions of containing a Buforinin, Previn, or other compound havingthe core structure of the invention. The compounds of the inventioncould be applied as a spray, foam, towelette, or sponge used to soak orwipe the surface. The amounts would be typically 200 ug per ml ofsolution applied; however, the concentrations required would depend onthe extent of contamination and the appropriate concentration of theactive ingredients may be adjusted as needed.

EXAMPLE 8 Prophylaxis Uses

Buforinins, Previns, and other compounds having the core sequence of theinvention could be used as a prophylactic against BttxB or Tttxpoisoning. Subjects could be treated with Buforinins, Previns, and othercompounds having the core sequence of the invention prior to enteringsituations where they are likely to be in contact with BttxB or Tttx.The dosage mode and amount could be dependent on the amount of toxinexpected to contact and the time in which contact might occur. Thepreferred administration for immediate contact would be i.v. Thepreferred form administration for a slower and more prolonged exposurewould be by ingestion. However, other slow release forms of deliverysuch as a patch may be used.

EXAMPLE 9 Prevention of Aerosol Contamination

Buforinins, Previns, and other compounds having the core sequence of theinvention may be incorporated into a disposable, moist-filter, breathingmask for inactivating BttxB in aerosol form. The toxin would be trappedin moist-filter whereupon it would inactivated by a Buforinin.

Such a filter design would protect against toxin particles smaller thanbacteria, e.g. 1 micron such as HEPA. The filters could be suppliedpremoistened and impregnated with Buforinins, Previns, or othercompounds having the core sequence of the invention and adjunctchemicals such as TCEP. Alternatively, the filters could be prepared bywetting a dry filter pre-impregnated or by soaking the filter in asolution of Buforinins, Previns, or other compounds having the coresequence of the invention. Enclosed areas that have air processingcapabilities may also be protected in this fashion with appropriatesized filters.

EXAMPLE 10 Wound Treatment

Open lesions could be treated with topical applications havingBuforinins, Previns, or other compounds having the core sequence of theinvention to inhibit BttxB or Tttx poisoning before the toxin has achance to be absorbed into the body. A powder mixture containingBuforinins and adjuncts which include a reducing agent and otherstabilizers or fillers may be applied directly to the wound. Thisapproach relies on the wound weeping to dissolve the Buforinins,Previns, or other compounds having the core sequence of the invention.Alternatively, an ointment, liquid, spray, foam, or towelette havingBuforinins, Previns, or other compounds having the core sequence of theinvention may be applied to the wound surface. The towelette could besupplied or made in a similar manner as the filters of Example 10.

EXAMPLE 11 Post Exposure

Subjects already suffering from BttxB or Tttx poisoning could be treatedwith Buforinins, Previns, or other compounds having the core sequence ofthe invention. These of treatments would scavenge accessible toxin notyet compartmentalized into susceptible cells. Intoxication ofsusceptible cells leads to cell function inhibition but is not itselflethal to the cells. Given sufficient time the cells can recover andbecome functional again. This recovery process may last up to severalmonths. Therefore, treatment with Buforinins, Previns, or othercompounds having the core sequence of the invention will aid in therecovery of the subject and reduce the need of alternative lifesupporting measures. The treatment may comprise use of Buforinin-BttxBHC or other Previns or other compounds having the core sequence of theinvention like conjugates. The Bttx-HC portion would specifically directthe conjugate to susceptible cells where uptake would occur in a mannersimilar as the toxin. Inside the cell, the conjugates would access tothe toxin and inhibit the protease activity, thereby protecting the cellagainst further toxin damage until the toxin is removed from the cellsby endogenous proteolysis.

EXAMPLE 12 Identification of a Botulinum Toxin Subclass

Buforinins, Previns, or other compounds having the core sequence of theinvention may be used for the identification of BttxB or Tttx. Anunknown Bttx or Tttx would be incubated with substrates and a Buforinin,Previn, or other compound having the core sequence of the invention thatwould specifically inhibit BttxB and Tttx if present. Detection ofuncleaved substrate or reduction of digest products would allow theidentification of the toxin.

This may be useful as a confirming assay since the inhibition isspecific. For example, a C-terminal fluorescent-labeled substrate, suchas VAMP2, would be attached to microtiter plates. See Hallis, B., et al.(1996) J. Clin. Microbiol. 34:1934-1938. The unknown sample is thenadded to the well and allowed to incubate. The reaction would be stoppedand the well rinsed. Reduction of fluorescence would indicatesusceptibility of the substrate to the toxin. If Buforinins, Previns, orother compounds having the core sequence of the invention are includedin the digest mix then BttxB or Tttx toxin would be specificallyinhibited and the fluorescence levels would be higher than thosereactions containing BttxB without inhibitor.

EXAMPLE 13 Construction of Compounds having Core Structures Optimizedfor Inhibiting the Protease Activity of Botulinum B toxin

Core structures of the formula (1), (2) or (3) may be optimized bymaking modifications or substitutions in the sequence such as thoseshown in FIG. 7. These core structures may then be used to constructcompounds which highly inhibit the protease activity of Botulinum Btoxin.

For example, by using standard methods and techniques, the corestructures of the invention may be modified by either making mutationsor substitutions and amino acid additions which favor turn formation.Other amino acid additions, mutations or substitutions could be made sothat either the helix becomes more amphipathic to improve helix bundlingor improve interaction with the toxin.

Incorporation by Reference

To the extent necessary to understand or complete the disclosure of thepresent invention, all publications, patents, and patent applicationsmentioned herein are expressly incorporated by reference therein to thesame extent as though each were individually so incorporated.

1-7. (canceled)
 8. A recombinant expression system for production of apeptide of claim 29 capable of inhibiting the protease activity ofBotulinum toxin B or tetanus toxin which expression system comprises anucleotide sequence encoding said peptide operably linked to a controlsequence for effecting expression.
 9. The recombinant expression systemof claim 8 wherein the nucleotide sequence encoding said peptide encodesa precursor peptide.
 10. A recombinant host cell modified to contain theexpression system of claim
 8. 11. A method to produce a peptide capableof inhibiting the protease activity of Botulinum toxin B or tetanustoxin which method comprises culturing the modified host cells of claim10 under conditions wherein said peptide is produced. 12-15. (canceled)16. A pharmaceutical composition for treating Botulinum or tetanusintoxication which comprises a protease inhibitory amount of a peptideselected from the group consisting of RPKPWWFFGLM, (SEQ ID NO: 1)TRSSRAGLQFPVGRVHRLLRK, (SEQ ID NO: 2) TRAARAGLQFPVGRVHRLLRK, (SEQ ID NO:3) TRLLRAGLQFPVGRVHRLLRK, (SEQ ID No: 4) RAKAQQFFGLM, (SEQ ID NO: 6)RAKAQQFPGLM, (SEQ ID NO: 7) RAKLQQFPGLM, (SEQ ID NO: 8) RAKGLQFPGLM,(SEQ ID NO: 9) RAGLGQFFGLM, (SEQ ID NO: 10) DAARAKGLQFPGLMAKLK, (SEQ IDNO: 11) DAARAKGLQFPGLLAKLK, (SEQ ID NO: 12) and TRSRAKGLQFPGLMVHRL, (SEQID NO: 13)


17. Antibodies specifically immunoreactive with the peptide of claim 29.18. An assay comprising contacting a sample suspected of containing aPrevin with the antibodies of claim 17 under conditions where theantibody would bind the Previn and detecting the bound Previn. 19-23.(canceled)
 24. A method for treating Botulinum or tetanus intoxicationcomprising administering to a subject, suspected of having Botulinum ortetanus intoxication or being in the presence of a causative agentthereof a protease inhibitory amount of a peptide or salts, esters,amides, and acyl forms thereof, wherein said peptide is selected fromthe group consisting of RPKPQQFFGLM, (SEQ ID NO:1)TRSSRAGLQFPVGRVHRLLRK, (SEQ ID NO:2) TRAARAGLQFPVGRVHRLLRK, (SEQ IDNO:3) TRLLRAGLQFPVGRVHRLLRK, (SEQ ID NO:4) RAKAQQFFGLM, (SEQ ID NO:6)RAKAQQFPGLM, (SEQ ID NO:7) RAKLQQFPGLM, (SEQ ID NO:8) RAKGLQFPGLM, (SEQID NO:9) RAGLGQFFGLM, (SEQ ID NO:10) DAARAKGLQFPGLMAKLK, (SEQ ID NO:11)DAARAKGLQFPGLLAKLK, (SEQ ID NO:12) and TRSRAKGLQFPGLMVHRL, (SEQ IDNO:13)


25. A kit for treating or preventing Botulinum toxin B or Tetanus toxinintoxication comprising at least one peptide of claim
 29. 26. A kit fordetermining whether a sample contains a Previn comprising antibodies ofclaim
 17. 27. (canceled)
 28. (canceled)
 29. A peptide in purified andisolated form, or salts, esters, amides and acyl forms thereof, selectedfrom the group consisting of RPKPQQFFGLM, (SEQ ID NO:1)TRSSRAGLQFPVGRVHRLLRK, (SEQ ID NO:2) TRAARAGLQFPVGRVHRLLRK, (SEQ IDNO:3) TRLLRAGLQFPVGRVHRLLRK, (SEQ ID NO:4) RAKAQQFFGLM, (SEQ ID NO:6)RAKAQQFPGLM, (SEQ ID NO:7) RAKLQQFPGLM, (SEQ ID NO:8) RAKGLQFPGLM, (SEQID NO:9) RAGLGQFFGLM, (SEQ ID NO:10) DAARAKGLQFPGLMAKLK, (SEQ ID NO:11)DAARAKGLQFPGLLAKLK, (SEQ ID NO:12) TRSRAKGLQFPGLMVHRL. (SEQ ID NO:13)


30. The peptide of claim 29, wherein the salt is a phosphate.
 31. Thepeptide of claim 30, wherein the phosphate salt is formed byphosphorylation of S, T, and/or Y.
 32. The peptide of claim 31, whereinthe peptide is characterized as having an improved circulatoryhalf-life, solubility, resistance to degradation, and interaction withthe active site of the toxin.
 33. The pharmaceutical composition ofclaim 16, wherein the salt is a phosphate.
 34. The pharmaceuticalcomposition claim 33, wherein the phosphate salt is formed byphosphorylation of S, T and/or Y.
 35. The pharmaceutical composition ofclaim 34, wherein the compound is characterized as having an improvedcirculatory half-life, solubility, resistance to degradation, andinteraction with the active site of the toxin.
 36. The pharmaceuticalcomposition of claim 16 further comprisingtris-(2-carboxyethyl)phosphine (TCEP).
 37. The pharmaceuticalcomposition of claim 36 further comprising biocompatable chaotropes. 38.The pharmaceutical composition of claim 37, wherein the biocompatiblechaotropes is hydroxyurea or 2-oxo-1 pyridine acetamide.
 39. The methodof claim 24, wherein the composition is administered to the subjectprior to the subjects contact with Botulinum or tetanus intoxication.40. The method of claim 39, wherein the contact is through aerosolcontamination.
 41. The method of claim 40, wherein the administrationinvolves impregnation of a filter with the compound.
 42. The method ofclaim 41, wherein the filter is a breathing filter affixed to thesubject after impregnation of the filter.
 43. The method of claim 24,wherein the peptide is administered directly to a wound on the subject.44. The method of claim 24, wherein the peptide is conjugated to Bttx-HCwhich directs the compounds to the desired site upon administration.